Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
Volume 12, Number 10—October 2006

Human Rotavirus G9 and G3 as Major Cause of Diarrhea in Hospitalized Children, Spain

Article Metrics
citations of this article
EID Journal Metrics on Scopus
Alicia Sánchez-Fauquier*Comments to Author , Vanessa Montero*, Silvia Moreno*, Monica Solé*, Javier Colomina†, Miren Iturriza-Gomara‡, Ana Revilla*, Isabel Wilhelmi§, Jim Gray†, and Gegavi/VIGESS-Net Group
Author affiliations: *Instituto de Salud Carlos III, Majadahonda, Madrid, Spain; †Hospital La Ribera, Alzira, Valencia, Spain; ‡Centre for Infections Health Protection Agency, London, UK; §Hospital Severo Ochoa, Majadahonda, Madrid, Spain

Cite This Article


In Spain, diarrhea remains a major cause of illness among infants and young children. To determine the prevalence of rotavirus genotypes and temporal and geographic differences in strain distribution, a structured surveillance study of hospitalized children <5 years of age with diarrhea was initiated in different regions of Spain during 2005. Rotavirus was detected alone in samples from 362 (55.2%) samples and as a coinfection with other viruses in 41 samples (6.3%). Enteropathogenic bacterial agents were detected in 4.9% of samples; astrovirus and norovirus RNA was detected in 3.2% and 12.0% samples, respectively; and adenovirus antigen was detected in 1.8% samples. Including mixed infections, the most predominant G type was G9 (50.6%), followed by G3 (33.0%) and G1 (20.2%). Infection with multiple rotavirus strains was detected in >11.4% of the samples studied during 2005.

Group A rotaviruses are a major cause of severe diarrhea in infants. In developing countries, severe diarrhea caused by human rotavirus results in an estimated 500,000 to 608,000 childhood deaths annually; worldwide, it results in ≈2 million hospitalizations (1,2).

Rotaviruses belong to the Reoviridae family. Viral particles are nonenveloped, and triple-layered protein capsids enclose the genome of 11 dsRNA segments. The major protein in the central layer of the viral capsid is VP6, which determines 7 different groups of rotaviruses (A–G). The outer layer of the viral capsid is composed of 2 structural proteins, VP4 (encoded by gene 4) and VP7 (encoded by gene 7, 8, or 9, depending on the strain) (3). These 2 proteins carry the major antigenic determinants, which elicit neutralizing antibodies and are thought to be type specific. Group A rotaviruses are widespread in humans and animals and are subdivided into distinct genotypes, G and P (4). Epidemiologic studies of rotavirus infections are increasingly showing that a great diversity of rotavirus strains are cocirculating in the human population throughout the world. The most common genotypes of group A rotaviruses (≈90%), which cause dehydrating gastroenteritis in infants and young children worldwide, were G1P[8], G2P[4], G3P[8], and G4P[8]; G1P[8] is the most prevalent worldwide (5). However, other G genotypes are epidemiologically important, such as G5 in Brazil (6,7), G9 and G10 in India (8,9), and G8 in Malawi (10).

In Spain, diarrhea remains an important cause of illness among infants and young children. A study conducted from 1998 through 2002 detected rotavirus in 1,155 (31%) of 3,760 specimens tested. G1 was the predominant genotype detected (53%), followed by G4 (24%), G2 (14%), G9 (6%), and G3 (2%) (11). The distribution of genotypes indicated a genotypic shift over time: G4 strains predominated (57%) from 1998 through 2000, whereas G1 gradually increased to account for 75% from 2000 through 2002 (11). Similar studies conducted in other regions of Spain indicated similar shifts in the prevalence of rotavirus genotypes (12,13).

We conducted structured surveillance among children with diarrhea who were hospitalized in 6 hospitals in Spain; our primary goals were to determine the prevalence of rotavirus diarrhea in hospitalized children, the G and P types among infecting rotavirus strains, and the temporal and geographic differences in strain distribution throughout the regions.

Materials and Methods

Hospitals and Patients

Stool samples were collected from children attending 6 public hospitals located in different healthcare areas throughout Spain. These hospitals intentionally represented the geographic, climatic, and ethnic diversity of Spain. Their respective catchment areas are shown in Table 1. The study was conducted between January 2005 and January 2006 and included children <5years of age who were hospitalized with acute gastroenteritis and from whom a stool sample was obtained.

Acute gastroenteritis was defined as >3 looser-than-normal stools within a 24-hour period or an episode of forceful vomiting and any loose stool. To enable reporting of test results to hospitals, stool specimens were labeled with the date of collection and a unique surveillance identification number. Permission for enrollment in the study was obtained from children's legal guardians, and ethical approval was obtained from the institutional review board of the Hospital de La Ribera.

Specimen Collection and Testing

Whole stool specimens were collected and transported immediately to hospital laboratories and stored at 4°C until processing. All fecal samples were screened for enteropathogenic bacterial agents by conventional culture methods previously described (14).

Each month, specimens were sent to the reference laboratory (Viral Gastroenteritis Unit, National Center for Microbiology, Instituto de Salud Carlos III, Madrid, Spain). A 10% suspension in 0.1 mol/L phosphate-buffered saline (pH 7.2) was prepared and tested by reverse transcription (RT)-PCR for rotavirus, astrovirus, norovirus, and sapovirus (11,15,16) and by an immunochromatographic method for enteric adenoviruses (14).

Nucleic Acid Extraction and G/P Rotavirus Typing

Viral RNA was extracted from 250 μL of the 10% fecal suspension by using the guanidine isothiocyanate method and the Rnaid Spin Kit (BIO 101, Anachem Bioscience, Bedfordshire, UK) according to the manufacturer's instructions, with slight modifications (16). RNA was eluted in 50 μL of RNase-free distilled water and stored at –20°C. To determine the G/P type patterns present in children hospitalized from 2005 through 2006, a total of 98 rotavirus strains were P typed. G and P rotavirus genotyping were performed by using RT-PCR methods as previously reported (11,17).

DNA Sequencing and Analysis

Rotavirus amplicons were genetically characterized by nucleotide sequencing of both strands of the amplified PCR products. These products were purified by using QIAquick PCR Purification kit (Qiagen, Valencia, CA, USA) and then sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on an ABI automated sequencer (Applied Biosystems, model 3700). Data analysis was performed by using Clustal for multiple alignments and neighbor-joining and maximum parsimony methods for phylogenetic analysis (Bionumerics, Kortrijk, Belgium). Spanish strains were submitted to GenBank under accession numbers DQ440613 through DQ440624.



A total of 656 hospitalized children were enrolled. Enteropathogenic bacterial strains were detected in 5.0% of samples (Table 2). Astrovirus and norovirus RNA was detected in 3.2% and 12.0% samples, respectively, and adenovirus antigen in 1.8% samples.

A total of 403 rotavirus strains were detected. Rotavirus was found alone in 362 (55.2%) samples but was found in another 41 samples (6.3%) as a coinfection with other viruses. The percentage of children with gastroenteritis caused by rotavirus as unique agent ranged from 36.7% in Leon to 68.2% in Valencia (Table 2).

Rotavirus Characterization

G typing RT-PCR for rotavirus alone was performed on 362 samples positive for rotavirus but could not be determined in 10 (2.8%) samples. The G types detected, including mixed infections with multiple rotavirus strains, are shown in Table 3. Briefly, the most predominant G type was G9 (50.6%), followed by G3 (33.0%), G1 (20.2%), and G2 (7.1%); the least common G type was G4 (0.6%). G1, previously reported as the most common G type in Spain, was found in only 20.2% of rotavirus infections. With the exceptions of Valencia and Albacete, where G1 and G3, respectively, were the predominant G types, the results from all other regions showed a predominance of G9. However, even in these 2 areas, G9 was the second most common strain detected when cases with coinfection were added (26.7% and 31.6%, respectively).

Common G/P combinations, infrequent patterns, and mixed-infection combinations were all detected (Table 4). G9P[8] (40%) and G3P[8] (31%) were the most common combinations detected, but G types in combination with P[6] and P[9] were also detected.

Using DNA sequencing and phylogenetic analysis of partial sequences of the gene encoding VP7, we compared 2 G3 strains from this study with 9 G3 strains isolated previously in Spain. All G3 strains from Spain shared >99.0% homology and were more closely related to each other than to strains isolated in Italy, United Kingdom, India, and China.


Genetically and antigenically diverse rotavirus strains cocirculate in humans. The prevalence of rotavirus genotypes varies according to location and time. Throughout the world, genotyping and serotyping studies have identified common cocirculating rotavirus types, and G1P[8], G2P[4], G3P[8], and G4P[8] are the predominant strains. However, from time to time, other less common genotypes, such as G9P[8], G5P[8], and G8P[6], have been predominant in various countries (5).

In Spain, previous studies have identified G1P[8] and G4P[8] as the predominant cocirculating strains from 1996 through 2004 (11,17,18) (Table 5). However, in our study, conducted in 2005 and 2006, a major shift in the predominant strains was detected. G9P[8] and G3P[8] have become the predominant genotypes cocirculating in several regions of Spain, and infection with multiple rotavirus strains was detected in 11.4% of the cases studied.

Since its widespread introduction into the human population in 1995, G9P[8] has become one of the predominant viruses worldwide. In 2 separate studies conducted in Thailand (19,20), this genotype has been reported as the predominant virus circulating from 2000 through 2002 and in Brazil from 1999 through 2002 (21). G3P[8] has recently been reported as the predominant strain circulating in the Japanese population (22).

Less common G- and P-type combinations were also detected in this study. This finding may suggest either an earlier reassortment between animal and human strains, resulting in the emergence of strains such as G2P[6] and G3P[9], or zoonotic transmission to humans of an animal strain, as possibly occurred with G9P[6]. The VP4-genotypes P[6] and P[9] are reported to be associated with infection in pigs and cats, respectively. Although animal rotavirus strains replicate poorly in humans and person-to-person transmission is rare, the relatively high frequency of multiple infections detected in this study suggests that the opportunity for dual infection of a cell, and therefore reassortments, exists (23).

The main limitations of this study are having only 1 year of data, the minimal variations in the sampling schemes in each institution (frequency of sampling, test procedures, motivations of investigators), and the small sample size collected. Although the sampling strategy enabled monitoring for rotavirus in a large number of children, future studies with hospital-based surveillance should be initiated in different areas of Spain, and even Europe, with larger samples.

Morbidity rates worldwide and morbidity and mortality rates caused by diarrhea in developing countries remain high despite efforts to improve sanitary conditions, water quality, and the healthcare infrastructure. These high rates have driven efforts to develop a safe and effective rotavirus vaccine, and the World Health Organization has recognized that developing a vaccine is a priority for reducing infant deaths in developing countries. The level and type of protection in rotavirus disease is poorly understood, although neutralizing antibody responses are thought to be type specific. Because these responses are associated with VP7 and VP4 viral proteins, establishing the G and P genotypes of strains circulating in the human population is important. Currently, 2 candidate rotavirus vaccines are undergoing clinical trials. A multivalent vaccine directed against G1, G2, G3, G4, and P[8] and a monovalent vaccine to G1P[8] have been developed (24,25). Homotypic protection has been demonstrated for both vaccines, but the degree to which they cross-protect against less common G- and P-type combinations not included in the vaccine formulations has yet to be established, and the importance of genotype-specific protection against rotavirus disease is still under discussion (26,27). Considering that G9 rotavirus type has emerged as one of the most common rotavirus genotypes in humans around the world, and it is becoming very prevalent in some countries, future rotavirus vaccine candidates will need to provide adequate protection against disease caused by G9 viruses. Therefore, surveillance of regional networks must be maintained to document rotavirus strain distribution and prevent the appearance of new strains or new variants that could escape immune protection induced by an outdated vaccine.

Dr Sánchez-Fauquier is the head of the Viral Gastroenteritis Unit, National Center for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid. Her primary research interests are the epidemiology, immunology, pathogenesis, and molecular biology of viral gastroenteritis. She also is coordinator of the Spanish Viral Gastroenteritis Network (VIGESS-Net).



We thank A. Potente for technical assistance and J.C. Sanz for fruitful discussions.

This work was partly supported by grant no. MPY1176/04 from ISCIII. V. Montero and S. Moreno were supported by a grant from ISCIII.



  1. Parashar  UD, Gibson  CJ, Bresse  JS, Glass  RI. Rotavirus and severe childhood diarrhea. Emerg Infect Dis. 2006;12:3046.PubMedGoogle Scholar
  2. Glass  RI, Bresee  JS, Parashar  U, Turcios  R, Fischer  TK, Jiang  B, Rotavirus vaccines: past, present, and future. Arch Pediatr. 2005;12:8447. DOIPubMedGoogle Scholar
  3. Kapikian  AZ, Hoshino  Y, Chanock  RM. Rotaviruses. In: Knipe DM, Howley, PM, editors. Fields virology. Philadelphia: Lippincott Williams and Wilkins; 2001. p. 1787–833.
  4. Desselberger  U, Wolleswinkel-van den Bosch  J, Mrukowicz  J, Rodrigo  C, Giaquinto  C, Vesikari  T. Rotavirus types in Europe and their significance for vaccination. Pediatr Infect Dis J. 2006;25(Suppl):S3041. DOIPubMedGoogle Scholar
  5. Santos  N, Hoshino  Y. Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev Med Virol. 2005;15:2956. DOIPubMedGoogle Scholar
  6. Gouvea  V, de Castro  L, Timenetsky  MC, Greenberg  H, Santos  N. Rotavirus serotype G5 associated with diarrhea in Brazilian children. J Clin Microbiol. 1994;32:14089.PubMedGoogle Scholar
  7. Leite  JP, Alfieri  AA, Woods  PA, Glass  RI, Gentsch  JR. Rotavirus G and P types circulating in Brazil: characterization by RT-PCR, probe hybridization, and sequence analysis. Arch Virol. 1996;141:236574. DOIPubMedGoogle Scholar
  8. Ramachandran  M, Das  BK, Vij  A, Kumar  R, Bhambal  SS, Kesari  N, Unusual diversity of human rotavirus G and P genotypes in India. J Clin Microbiol. 1996;34:4369.PubMedGoogle Scholar
  9. Iturriza Gomara  M, Kang  G, Mammen  A, Jana  AK, Abraham  M, Desselberger  U, Characterization of G10P[11] rotaviruses causing acute gastroenteritis in neonates and infants in Vellore, India. J Clin Microbiol. 2004;42:25417. DOIPubMedGoogle Scholar
  10. Cunliffe  NA, Gondwe  JS, Broadhead  RL, Molyneux  ME, Woods  PA, Bresee  JS, Rotavirus G and P types in children with acute diarrhea in Blantyre, Malawi, from 1997 to 1998: predominance of novel P[6]G8 strains. J Med Virol. 1999;57:30812. DOIPubMedGoogle Scholar
  11. Sánchez-Fauquier  A, Wilhelmi  I, Colomina  J, Cubero  E, Roman  E. Diversity of group A human rotavirus types circulating over a 4-year period in Madrid, Spain. J Clin Microbiol. 2004;42:160913. DOIPubMedGoogle Scholar
  12. Buesa  J, de Souza  CO, Asensi  M, Martinez  C, Prat  J, Gil  MT. VP7 and VP4 genotypes among rotavirus strains recovered from children with gastroenteritis over a 3-year period in Valencia, Spain. Eur J Epidemiol. 2000;16:5016. DOIPubMedGoogle Scholar
  13. Cilla  G, Perez-Trallero  E, Lopez-Lopategui  MC, Gilsetas  A, Gomariz  M. Incidence, seasonality and serotypes of rotavirus in Gipuzkoa (Basque Country), Spain. A 14-year study. Epidemiol Infect. 2000;125:67783. DOIPubMedGoogle Scholar
  14. Roman  E, Wilhelmi  I, Colomina  J, Villar  J, Cilleruelo  ML, Nebreda  V, Acute viral gastroenteritis: proportion and clinical relevance of multiple infections in Spanish children. J Med Microbiol. 2003;52:43540. DOIPubMedGoogle Scholar
  15. Dalton  RM, Roman  ER, Negredo  AA, Wilhelmi  ID, Glass  RI, Sánchez-Fauquier  A. Astrovirus acute gastroenteritis among children in Madrid, Spain. Pediatr Infect Dis J. 2002;21:103841. DOIPubMedGoogle Scholar
  16. Sánchez-Fauquier  A, Wilhelmi  I, Roman  E, Colomina  J, Montero  V, Negredo  A. Surveillance of human calicivirus in Spain. Emerg Infect Dis. 2005;11:13279.PubMedGoogle Scholar
  17. Wilhelmi  I, Mier  C, Roman  E, Colomina  J, Prat  J, Sánchez-Fauquier  A. The molecular epidemiology of the rotavirus in Spanish children: The Rotavirus Study Group (GER). Enferm Infecc Microbiol Clin. 1999;17:50914.PubMedGoogle Scholar
  18. Sánchez-Fauquier  A. Changing epidemiology of rotavirus diarrhea in Spain. 1st European Rotavirus Biology Meeting; 2005 Apr 24–26; Paris, France.
  19. Zhou  Y, Supawadee  J, Khamwan  C, Tonusin  S, Peerakome  S, Kim  B, Characterization of human rotavirus serotype G9 isolated in Japan and Thailand from 1995 to 1997. J Med Virol. 2001;65:61928. DOIPubMedGoogle Scholar
  20. Khamrin  P, Peerakome  S, Wongsawasdi  L, Tonusin  S, Sornchai  P, Maneerat  V, Emergence of human G9 rotavirus with an exceptionally high frequency in children admitted to hospital with diarrhea in Chiang Mai, Thailand. J Med Virol. 2006;78:27380. DOIPubMedGoogle Scholar
  21. Santos  N, Volotao  EM, Soares  CC, Campos  GS, Sardi  SI, Hoshino  Y. Predominance of rotavirus genotype G9 during the 1999, 2000, and 2002 seasons among hospitalized children in the city of Salvador, Bahia, Brazil: implications for future vaccine strategies. J Clin Microbiol. 2005;43:40649. DOIPubMedGoogle Scholar
  22. Zhou  Y, Li  L, Okitsu  S, Maneekarn  N, Ushijima  H. Distribution of human rotaviruses, especially G9 strains, in Japan from 1996 to 2000. Microbiol Immunol. 2003;47:5919.PubMedGoogle Scholar
  23. Iturriza-Gomara  M, Kang  G, Gray  J. Rotavirus genotyping: keeping up with an evolving population of human rotaviruses. J Clin Virol. 2004;31:25965. DOIPubMedGoogle Scholar
  24. Vesikari  T, Matson  DO, Dennehy  P, Van Damme  P, Santosham  M, Rodriguez  Z, Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med. 2006;354:2333. DOIPubMedGoogle Scholar
  25. Ruiz-Palacios  GM, Perez-Schael  I, Velazquez  FR, Abate  H, Breuer  T, Clemens  SC, Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis. N Engl J Med. 2006;354:1122. DOIPubMedGoogle Scholar
  26. Hoshino  Y, Kapikian  AZ. Rotavirus serotypes: classification and importance in epidemiology, immunity, and vaccine development. J Health Popul Nutr. 2000;18:514.PubMedGoogle Scholar
  27. Jiang  B, Gentsch  JR, Glass  R. The role of serum antibodies in the protection against rotavirus disease: an overview. Clin Infect Dis. 2002;34:135161. DOIPubMedGoogle Scholar




Cite This Article

DOI: 10.3201/eid1210.060384

1Gegavi/VIGESS-Net Group members: A. Sánchez-Fauquier, V. Montero, S. Moreno, A. Potente, F. Adam, J.C. Sanz, J. Colomina, S. Llanes, F. Gimeno, C. Gutiérrez, C. Sainz de Baranda, M.J. López, P. Teno, E. Roman, M. Alonso, M. Marugán, I. Fernández, I. Wilhelmi, M.L. Cilleruelo

Table of Contents – Volume 12, Number 10—October 2006

EID Search Options
presentation_01 Advanced Article Search – Search articles by author and/or keyword.
presentation_01 Articles by Country Search – Search articles by the topic country.
presentation_01 Article Type Search – Search articles by article type and issue.



Please use the form below to submit correspondence to the authors or contact them at the following address:

Alicia Sánchez-Fauquier, Sección de Gastroenteritis Virales, Centro Nacional de Microbiología, Instituto Salud Carlos III, Ctra. Majadahonda-Pozuelo Km. 2, 28220-Majadahonda, Madrid, Spain

Send To

10000 character(s) remaining.


Page created: November 10, 2011
Page updated: November 10, 2011
Page reviewed: November 10, 2011
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.